Proteomic Analysis of the Effect of Fuzheng Huayu Recipe on Fibrotic Liver in Rats

Hepatic fibrosis is a common pathological process of chronic liver diseases and would lead to cirrhosis, and Fuzheng Huayu (FZHY) is an effective Chinese herbal product against liver fibrosis. This study observes FZHY influence on proteome of fibrotic liver with differential proteomic approach and aims to understand FZHY multiple action mechanisms on liver fibrosis. The liver fibrosis models were induced with intraperitoneal injection of dimethylnitrosamine for 4 weeks in rats and divided into model control (model) and FZHY-treated (FZHY) groups, while normal rats were used as normal control (normal). After model establishment, rats in FZHY groups were administered 4 g/kg wt of FZHY for 4 weeks, and normal and model groups were given the same volume of saline. The liver proteins in the above 3 groups were separated by two-dimensional gel electrophoresis (2-DE), the differentially expressed spots were analyzed and compared between normal and model or model and FZHY groups, and then the proteins were identified with mass spectrum analysis and validated partially with western blot and real-time PCR. 1000~1200 spots were displayed on each 2D gel, and a total of 61 protein spots were found with significant intensity difference between normal control or FZHY and model control. 23 most obviously differential spots were excised, and in-gel digestion and 21 peptide mass fingerprints (PMF) were obtained with MALDI-TOF MS analysis, and 14 proteins were identified through protein database searching. Among 14 differentially expressed proteins, 8 proteins in normal and FZHY groups had the same tendency of differential expression compared with the ones in model group. And one of them, vimentin, was validated by western blot and real-time PCR analyses. Our study reveals 12 proteins responsible for fibrogenesis induced by DMN in rats, and among them, 8 proteins in fibrotic liver were regulated by FZHY, including aldehyde dehydrogenase, vimentin isoform (CRA_b), gamma-actin, vimentin, fructose-bisphosphate aldolase B, aldo-keto reductase, S-adenosylhomocysteine hydrolase isoform, and HSP90. It indicates that the action mechanism of FZHY antiliver fibrosis may be associated with modulation of proteins associated with metabolism and stress response, as well as myofibroblast activation. The study provides new insights and data for exploring the liver fibrogenesis pathophysiology and FZHY action mechanism against liver fibrosis.


Introduction
Liver �brosis and cirrhosis represent the conse�uences of a sustained wound healing response to chronic liver injury from a variety of causes, including viral, autoimmune, druginduced, cholestatic, alcoholic, and metabolic diseases [1]. e matrix component of the scar tissue in cirrhosis is similar regardless of its etiologies [2]. ese scar constituents accumulate from a net increase in liver extracellular matrix (ECM) components, regulated mainly by hepatic stellate cells (HSCs), which are mediated by various cytokines, growth factors, and proteases and their inhibitors [3,4]. Recent decades had witnessed the signi�cant progress in the understanding of liver injury and �brosis; however the e�cient, and welltolerated anti�brotic drugs are still missing, the mechanisms of liver �brogenesis is not fully elucidated and speci�c anti-�brotic drug targets are still lacking [5]. erefore, it is very important to further explore the molecular mechanism 2 Evidence-Based Complementary and Alternative Medicine of liver �brosis, in particular, to seek effective and safe medicines for liver �brosis treatment.
ere is a recent increasing interest in Traditional Chinese medicine (TCM) or other botanic medicines, which have been used for thousands of years because of their clinic efficacy and easy applicability, in particular in the �eld of liver diseases. However, TCM usually consists of complex mixtures and compositions of herbs and apparently exerts its action through multiple pathways [6]. It is difficult to understand the complicated action mechanisms of TCMs fully with conventional methodologies such as western blot analysis, which can semiquantitatively determine the expression of proteins of interest [7]. Proteomics and other system biology approaches could simultaneously generate large biological data sets and provide powerful tools for the understanding of the mechanisms of TCM [8]. Especially, the comparison of the expressional proteome between normal and diseased sample (cells or tissue) or between the diseased and treated sample would be very helpful to explore disease-or drug-speci�c differential proteome and can lead to identify the molecular targets involved in different pathophysiological states of the diseases and to understand the complex action mechanisms of medicines including TCM.
Fuzheng Huayu recipe (FZHY), a prescription usually used for treating liver �brosis in traditional Chinese medicine (TCM), is made of six traditional Chinese drugs: Radix Salvia Miltiorrhizae (Danshen), Cordyceps (Chongcao), Semen Persicae, Gynostemma Pentaphyllammak (Jiaogulan), Pollen Pini (Song hua fen), and Fructus Schisandrae Chinensis (Wuweizi) [9]. Our previous studies suggested that the recipe could signi�cantly alleviate liver �brosis in animal models through anti-in�ammation, antioxidative stress, antiproliferation, and activation of hepatic stellate cells (HSCs), protection of liver function, decreasing the collagen synthesis and promoting degradation of extracellular matrices (ECM) [10][11][12][13][14][15]. Additionally, a multicenter, randomized, double-blinded, and parallel control experiment demonstrated that FZHY had good therapeutic effects on improving liver �brosis due to chronic hepatitis B [16]. e six herbs containing recipe are complex mixtures of ingredients, which act in concert to treat imbalanced body symptoms, likely with the mechanisms of simultaneously treating multiple therapeutic targets [9]. Albeit a great deal has been done to understand the therapeutic mechanism of the detailed mechanism is still unclear. In the present study we used contemporary proteomics tools to compare the differences in protein patterns of liver from normal, DMN-induced �brotic rats and FZHY-treated rats. Furthermore, the action mechanism of FZHY anti�brosis of liver is discussed.  Table 2). In the present study, FZHY powder was suspended in distilled water at a concentration of 0.5 g/mL for administration to animals. FZHY was administrated daily by intragastric gavage at a dose of 4.0 g (crude drug)/kg body weight.

Animals.
Male SD rats of weighting 120-150 g were used. e rats were fed with standard rat diet and water according to the guidelines approved by the Chinese Association of Laboratory Animal Care. e liver �brosis models were induced with intraperitoneal injection of dimethylnitrosamine (DMN, Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) at a dosage of 10 g/kg body wt for consecutive 3 days weekly and totally for 4 weeks [17]. Aer DMN intoxication, model rats were divided into model control (model, ) and FZHY treated (FZHY, ) groups, while normal rats were used as normal control (normal, ). Aer model establishment, FZHY groups orally took 4 g/kg wt of FZHY for 4 weeks, and the normal and model control groups took the same volume of saline. At the end of the treatment, all rats were sacri�ced and their blood and liver tissues were collected. A portion of liver tissues was �xed in 10% phosphate-buffered formalin for histological studies aer paraffin embedding. e remainder was snap-frozen  27.3% (v/v) perchloric acid in isopropanol) at 50 ∘ C for 90 min and measured at A558 nM. All results were normalized by total protein concentration and calculated using a standard curve.

Tissue Specimen and Sample Preparation for 2DE
. ree liver samples were selected from each group and homogenized in liquid nitrogen-cooled mortar and pestle and then dissolved in lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris, 65 mM DTT). Samples were sonicated on ice for 10 sec, three times in an ultrasonic processor and centrifuged for 1 h at 20,627 ×g (15,000 RPM) to remove DNA, RNA, and any particulates. e concentrations of all samples were measured by a modi�ed Bradford assay (Bio-Rad). e extracts from the same group were pooled with equal amounts and the concentrations were measured again. All samples were stored at −80 ∘ C until further processed.

Two-Dimensional Electrophoresis (2DE) and
Image Analysis. 2DE and image analysis was performed according to previously described methods [18] with some modi�cations. Brie�y, the �rst-dimensional isoelectric focusing (IEF) step was accomplished on an IPGphor IEF system (Amersham Biosciences, Uppsala, Sweden). 100 g of total proteins for analytical or 1.0 mg for preparative runs were mixed with a rehydration solution (8 M Urea, 2% CHAPS, 18 mM DTT, 0.5% IPG buffer, and bromophenol blue) and applied to Immobiline pH-gradient IPG dry strips (IPG buffer, pH 3-10). Aer rehydration for 12 h in 250 L of rehydration buffer containing the protein samples, proteins were focused successively for 1 h at 500 V, 1 h at 1000 V, and 10 h at 8000 V on an IPGphor. Aer IEF, IPG strip was equilibrated for 2 × 15 min in 50 mM Tris-HCl, pH 8.0, 6 M urea, 30% glycerol, 2% SDS, and bromophenol blue containing buffer. DTT (1%) was added to the �rst equilibration buffer.
In the second equilibration buffer, DTT was replaced by 2.5% iodoacetamide (IAA), and the second dimension separation was performed with 12% sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in Ettan DALT II electrophoresis apparatus. e analytical gels were visualized with silver staining, while the preparative gels were stained with Coomassie Blue G250 (Bio-Rad). e silverstained 2-D gels were scanned at an optical resolution of 84.7 um/pixel using a GS-710 imaging densitometer (Bio-Rad). Spot detection, quanti�cation, and matching were performed using ImageMaster soware (GE healthcare, USA). Quantitative analysis was performed using the Student's ttest between normal and model groups or model and FZHY groups with a level of 95%.

In-Gel Digestion.
For MS �ngerprinting, gel plugs were cut out off the preparative Coomassie blue-stained gels, destained with 100 mM NH 4 HCO 3 in 30% acetonitrile ACN, and lyophilized (VirTis Vacuum-Spin, NY, USA). e dried gel plugs were rehydrated with a total of 25 L of sequencing grade, modi�ed trypsin (Promega, Madison, USA) in 100 mM ammonium bicarbonate at 47 ∘ C for 2 h. en 20 L of 50 mmol/L NH 4 HCO 3 , pH 8.3 was added, and the gel slices were incubated at 37 ∘ C for 12 h. e digestion buffer was removed and saved. e gel pieces were extracted with 200 L of 60% ACN/0.1% TFA for 15 min with sonication, and the supernatant was removed. e extraction was repeated twice more and the three extracts plus the �rst saved digestion buffer fraction were pooled and dried completely under vacuum. is in-gel digestion method was mainly performed according to the method described previously [19] with the modi�cations as described previously.  Table 3, which were designed and synthesized by Sangon Biotech Inc. (Shanghai, China). PCR mixtures contained 1 L cDNA, 10 L SYBR Premix Ex Taq (2x, Takara, Dalian, China), and 0.25 M forward and reverse primers in a �nal volume of 20 L. Triplicates were performed with a Rcorbett 6.0 system (Rotor-Gene 3000, Australia) starting with a polymerase activation step for 10 s at 95 ∘ C, followed by 40 cycles of 5 s at 95 ∘ C, 15 s at 58 ∘ C, and 10 s at 72 ∘ C. Fluorescence data were acquired aer each cycle. e absence of primer dimers and unspeci�c products was veri�ed aer every run by melting curve analysis (72 to 95 ∘ C) and agarose gel electrophoresis.

2.12.
Statistics. Date were expressed as mean ± SD. Statistical analysis was evaluated by one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test for multiple comparisons, which was used to evaluate the difference between two groups. was considered to be signi�cant. still developed severe hepatic injury in the liver, re�ected by the elevating ALT level compared to control rats; however, rats treated with FZHY showed a lower level, the albumin in DMN-induced rats decreased compared to control mice, while FZHY improved the albumin level (Figure 1(a)). As shown in Figure 1(b) by sirus red staining, model rats developed �brosis in the liver and FZHY administration greatly reduced accumulation of collagen in the tissue. Similarly, the Hyp content was signi�cantly greater in the model rats' liver compared to control rats. FZHY treatment, however, remarkably decreased the Hyp content in the livers of model rats (Figure 1(c)). ese �ndings show that FZHY exerted good e�ects ameliorating hepatic injuries and �brosis in DMN-induced rats, so next we were eager to know how FZHY did that.

Differential 2DE Analysis of Liver Tissue
Proteins. e 2DE gel shows a typical separation of liver tissue proteins (total) in normal, model, and FZHY groups into 1000∼ 1200 spots. e 2DE experiment was repeated three times. Nine 2DE gel images were analyzed, and one of the most reproducible images from model group tissue sample was selected as a reference gel. With ImageMaster soware, ratios of normalized spot intensities of normal to model control tissue or model to FZHY tissue were calculated. A total of 61 protein spots exhibited signi�cant intensity changes as the gels between normal and model or between model and FZHY were image analyzed and compared ( ) (Figure 2). We grouped these 61 differentially expressed protein spots into three major patterns ( Figure 3): pattern A: 54 spots differentially expressed between normal and model group; pattern B: 18 spots differentially expressed between model and FZHY group; pattern C ( Figure 4): 11 spots were overlapped between pattern A and pattern B, which means these 11 spots were differentially expressed among all 3 groups. More importantly, the 11 spots in the normal and FZHY groups showed the same differential expression compared with the model group. For example, when a spot intensity in the normal group was decreased compared to model, the corresponding spot intensity in FZHY also decreased.

�denti�cation of t�e Differentially Expressed Proteins by MS.
Among the previously identi�ed 61 differentially expressed protein spots, the 23 spots showing the largest difference were excised from the preparative gels, followed by in-gel tryptic digestion. 21 peptide mass �ngerprints (PMFs) were successfully identi�ed through analysis with MALDI-TOF MS and through protein database searching; 15 PMFs matched the database information and 6 PMFs failed. Interesting to note that three different spots (spots no. 1194, 1334, 1208) were identi�ed as vimentin, among them spots 1069 and spot 1194 were identi�ed as vimentin isoforms. erefore we actually obtained a total of 14 differentially expressed proteins, among them, 12 belonged to pattern A, 9 belonged to pattern B, while 8 proteins were overlapped between patterns A and B and belonged to pattern C. e protein IDs and descriptions are shown in Tables 4 and 5.

e Differentially Expressed Proteins between Normal and Fibrotic Liver.
Liver �brosis is orchestra of multiplex disorders involved in many liver cells and cytokines [20]. Although recent years have witnessed big progresses in understanding the mechanism of liver �brosis, including elucidating the pivotal role of hepatic stellate cell activation and transforming growth factor-1 in the formation of liver �brosis, the complicated mechanism of �brogenesis is not yet fully understood. e proteomics analysis of liver �brotic animals and cells could provide useful information for understanding liver �brogenesis, �nding potential diagnostic markers and discovering therapeutic target candidates [21][22][23][24][25][26]. In the present study, we used a 2DE-based proteomic approach to separate liver proteins and MALDI-TOF MS for their identi�cation in distinct proteomes of normal rat liver and �brotic liver. Our result showed that 12 proteins were expressed differentially between normal and �brotic (model) livers as listed in Table 4, which were mainly involved in �ve biological aspects, such as substance metabolism, protein binding, oxidative stress, stress response and cellular calcium ion homeostasis. Up-and downregulated proteins were classi�ed by the biological processes in which they were supposed to be involved according to gene ontology criteria (http://www.ebi.ac.uk/Databases/ontology.html). Compared to the normal liver, the �brotic liver had the decreased expression levels of catalase, clathrin light chain, regucalcin, fructose-bisphosphate aldolase B, and aldo-keto Differentially F 3: Grouping of differentially expressed protein spots. rough comparison of 2DE gels between two groups with a gel-in model as a reference, there were 54 differential spots between normal and model groups (pattern A), 18 differential between model and FZHY groups, and 11 spots were overlapped between pattern A and pattern B, in which normal and FZHY groups had the same tendency of differential expression compared with ones in model group (pattern C). reductase family 1, but increased levels of vimentin, Glutathione S-transferase, Aldehyde dehydrogenase, -actin and S-adenosylhomocysteine hydrolase, isoform, and so forth. Among these differential expressed proteins, aldehyde dehydrogenase [27], fructose-bisphosphate aldolase B [28], vimentin [29], heat shock protein 90- [21], catalase [30], and glutathione S-transferase [31] had already been described in the context of �brogenesis; others are not reported to have link with liver �brosis. Regucalcin was related to cellular calcium ion homeostasis, which was reportedly involved in  F 4: e differentially expressed 11 spots among all 3 groups on 2DE gel from a preparative gel. e 11 spots marked with red circles and ID codes expressed among all three groups. More importantly, the 11 spots in the normal and FZHY group showed the same differential expression compared with the model group.
1.0 mg of total protein was isoelectrically focused on IPG strips (pH 3-10), then separated by 12% SDS-PAGE as seconddimension. e protein spots were visualized by Coomassie Blue G250 staining.
chronic liver injury and acute liver failure [32][33][34] through regulating ATPase activity and calcium-mediated signaling, which may be involved in �brosis. �ther proteins, including aldo-keto reductase family 1, member D1, Sadenosylhomocysteine hydrolase, and clathrin light chain, even have not been reported to be linked to liver damage or cirrhosis.

e Differentially Expressed Proteins between Fibrotic
Liver and FZHY-Treated Liver and Validation by Western Blotting and Real-Time PCR. �e also identi�ed 9 differentially expressed proteins between the model and FZHY groups (Table 4). Among them, 8 proteins were also present as differentially expressed proteins between normal and model samples as shown in Table 5. More importantly, these 8 differentially expressed proteins among 3 groups as pattern C had very interesting feature; all of 8 proteins in normal and FZHY groups had the same tendency of differential expression compared with the ones in model group. For example, normal group had a very lower expression of vimentin, compared to model, however, and FZHY group had decreased vimentin level too. It indicates that FZHY could restore proteins expressions which were expressed abnormally in �brotic liver, and these differentially expressed proteins among 3 groups provide new insights into elucidation of FZHY action mechanism against liver �brosis.
To con�rm the previous presented results, vimentin, one of differentially expressed proteins was selected to be validated by western blotting and PCR. Figure 5 showed vimentin with higher expression in the model group but signi�cantly downregulated in the normal and FZHY groups in 2DE gels. As shown in Figure 6, the western blot results  of vimentin were consistent with results of the 2DE gels. To examine whether protein alterations observed by proteomic analysis correlate with the changes of the respective mRNAs at the transcription level, vimentin was also chosen for further validation by real-time PCR. As shown in Table 6, the expression of vimentin mRNA dramatically increased in model group ( ), while signi�cantly decreased in FZHY group ( ). Vimentin, as the ma�or intermediate �lament protein with function of skeleton organization, is not only a kind of matrix component which contributes to �brosis formation, but also a marker of mesenchymal cells [35,36]. e effector cell for liver �brogenesis is myo�broblast, which can come from hepatic stellate cell (HSC) activation and epithelial cells such as hepatocyte transformation through the process of epithelial-to-mesenchymal transition named as EMT [37]. HSC activation and epithelial cells EMT both increased the expression of vimentin dramatically [36,38]. erefore, in the study, FZHY inhibits the increase vimentin and its isoform expression, and it not only recon�rms the FZHY efficacy on liver �brosis [16], but also suggests that FZHY action mechanism is related to inhibiting HSC activation or EMT in liver cells. Among other 7 proteins regulated by FZHY, gamma-actin had similar function as vimentin. Aldehyde dehydrogenase 1 family member A1, fructose-bisphosphate aldolase B, aldo-keto reductase family 1 member D1, Sadenosylhomocysteine hydrolase isoform, and HSP90 are related to stress response and substance metabolism including retinoic acid, carbohydrate, and bile acid. In the study, FZHY could in�uence the oxidative stress in liver, which is consistent with our previous report [39]. However, it is the �rst time to know that FZHY can modulate the substance metabolism in �brotic liver and cells, which maybe is a new action mechanism of FZHY against liver �brosis. Chaperonin subunit 8 was only differentially expressed between model and FZHY groups, and the validation and signi�cance would need further exploration.
Although two-dimensional gel provides high-resolution separation, it has a number of shortcomings. It is difficult to identify proteins of certain types, in particular, proteins with low abundances, membrane protein, and proteins at extreme of molecular size, while mammalian tissue has complex and high dynamic abundance ranges of proteins, which increase the challenge for the effective detection of low-abundance proteins such as transcription factors and cytokines. Although we get some new and valuable insights in the mechanism of liver �brosis and FZHY anti�brotic action in the study, we failed to characterize those well-identi�ed proteins (e.g., TGF-, MMPs, and TIMPs) closely involved in �brogenesis. is remains a common theme among studies of the liver proteome and emphasizes the importance of implementing additional strategies (e.g., subcellular fractionation, glycoproteome, or cysteinyl subproteome enrichment) to reduce sample complexity, improve proteome coverage, and enhance the detection of low-abundance proteins important to the study of mechanism of liver �brosis and therapeutic drug target [40]. And of course we still need combine the conventional approaches such as western blot for investigating pharmacological mechanisms.

Concluding Remarks
e current study demonstrated that there are 12 proteins responsible for �brogenesis induced by DMN in rats� the roles of regucalcin, aldo-keto reductase family 1, member D1, Sadenosylhomocysteine hydrolase, clathryn light chain in the liver �brogenesis were not clear yet. Also among them, 8 proteins in �brotic liver were regulated by FZHY, including aldehyde dehydrogenase, vimentin isoform (CRA_b), gammaactin, vimentin, fructose-bisphosphate aldolase B, aldo-keto reductase, S-adenosylhomocysteine hydrolase isoform, and HSP90. It indicates that the action mechanism of FZHY antiliver �brosis may be associated with modulation of proteins associated with metabolism and stress response, as well as myo�broblast activation. e study provides new insights and data for exploring the liver �brogenesis pathophysiology and FZHY action mechanism against liver �brosis.

Con�ict of �nterests
ere is no �nancial�commercial con�ict of interests.